In view of the developing interest in the importance of the glycosylation of proteins, it has become essential to develop new methods for the study of the detailed structure of complex glycans.
The glycans of glycoproteins are typically either N-linked (attached to asparagine) or O-linked (attached to serine or threonine) [1]. In order to carry out structural analysis of glycoproteins, it is desirable to release the glycans from the protein backbone. To this end, several enzymatic and chemical methods are available. Enzymatic release of N-glycans can be achieved with peptide N-glycosidases and endoglycosidases [2], but relatively few enzymes are available for removal of O-glycans [3]. Chemical treatment with anhydrous hydrazine [4] has been used to remove glycans of both classes, and has been adapted, using milder conditions, for the selective removal of O-glycans [5]. The usual method for the release of O-glycans however is by digestion of the glycoprotein in mild alkali, usually in the presence of borohydride [6]. A significant disadvantage of this method is a reduction of the glycans to alditols and that the reaction is not truly specific for O-glycans as there is significant release of N-glycans due to the inclusion of borohydride.
To date, the most common method for immobilising reducing sugars has been reductive amination [7], whereby the sugars are first attached to polymer-bound primary amino groups by a labile glycosyl amine linkage which is then stabilised by reduction. This reduction is slow and the efficiency of binding is rather low when limited amounts of sugars are available. In recent variations on this approach, the glycosyl amine linkage is stabilised by acylation [8] or Amadori rearrangement [9].
There is a need for site specific identification and characterisation of protein associated glycosylation. Edman degradation is the ideal chemical method for identifying single sites of glycosylation in proteins. The predominant technology of absorption-phase Edman degradation, however, fails to extract the glycosylated amino acid for subsequent analysis. In contrast solid-phase Edman degradation extracts the N- and O-glycosylated amino acids in anhydrous trifluoroacetic acid and each glycosylated amino acid can be collected and subjected to carbohydrate analysis and/or modification (10). The harsh conditions of Edman degradation, however, result in the hydrolysis of sialic acid from the oligosaccharides (FIG. 1). In addition, unless the sialic acid is removed the sialylated oligosaccharide is attached to the solid-support during the immobilisation procedures (FIG. 2). Hence, there is a need to develop a method of modifying sialic acid so as to prevent its immobilisation during the covalent-coupling chemistry and to impart stability during the acid conversion of the sialylated thiazilinone to the thiohydantoin (FIG. 3).
There is a strong need to have simple experimental conditions for the specific release of O-linked glycans from glycoproteins, and for their isolation in an unreduced form suitable inter alia for chemical derivatisation, reductive incorporation of labels or the attachment of polymer supports.
The present inventors have developed chemical modification procedures for the analysis and manipulation of sugars (FIG. 4). It will be appreciated by one skilled in the art that the following disclosures can be applied to monosaccharide or oligosaccharide moieties.